Inhibitory Effects and Mechanism of the Combined Use of α-Helical Peptides HPRP-A1/HPRP-A2 and Chlorhexidine Acetate Against Bacterial and Fungal Biofilms

Drug resistance is an increasing problem in the treatment of vaginitis bacterial vaginosis and vulvovaginal candidiasis caused by biofilm-forming bacteria and fungi. In this study, the antimicrobial activities of the synergistic effects coordinated by antimicrobial peptides HPRP-A1, HPRP-A2, and chlorhexidine acetate (CHA), against biofilms caused by Gram-negative and Gram-positive bacteria, and one fungus Candida albicans , were investigated in vitro and in mouse and rat infection models. The minimal biofilm inhibitory concentration and the fractional biofilm inhibitory concentration of HPRP-A1, HPRP-A2 alone and in combination with CHA were determined by classical methods. The crystal violet staining assay was used to determine the biomass of agents on biofilms. The MTT assay was used to determine the metabolic activity of agents on biofilms. Different microscopy assays were used to observe the morphological characteristics of bacterial and fungal biofilms after drug treatments. The extracellular polysaccharide was detected by a phenol–sulfuric acid method. The combined uses of HPRP-A1, HPRP-A2 and CHA exerted strong synergistic effects to inhibit the formation of bacterial and fungal biofilms and disrupt the construction of biofilms in vitro and in vivo. In animal vaginitis models, the drug combination reduced 99.9% of the number of bacterial and fungal CFU, and showed significant therapeutic effects against biofilm vaginitis. The mechanism of membrane disruption coordinated by the combination of drugs depends not only on the inhibition of extracellular polysaccharides secreted by bacterial and fungal cells, but also on the ability of the peptides to penetrate the cell membrane.